Allicin (diallyl thiosulfinate) has been shown to elicit a marked pulmonary vasodepressor response and is widely regarded as one of the most important biologically active compounds produced by garlic (Allium sativum). We reported that long-term oral garlic feeding in rats was effective in preventing the development of hypoxic pulmonary hypertension. However, inability to accurately detect allicin in plasma following garlic ingestion and a lack of understanding of its pulmonary vasodepressor actions have slowed its development as a complementary therapeutic agent. Recently, we developed a simple HPLC method coupled to mass spectrometry (MS-MRM) to precisely identify and quantify allicin content in both aqueous and plasma samples. We found that allicin content, from either pure allicin, freshly crushed garlic or freeze-dried preparations, correlated directly with a vasorelaxation responses, which in part mediated via an increase in nitric oxide (NO) production. The goal of this proposal is to investigate the cellular and molecular mechanisms of garlic/allicin modulation of pulmonary vasoactivity and function. Our hypothesis is that garlic and its active metabolite, allicin, act either singularly via an activation of endothelial nitric oxide synthase (NOS) and an increase in NO production or in concert with a direct inhibition of vascular smooth muscle contraction to modulate pulmonary vascular tone and reactivity. Five Specific Aims are proposed. We will investigate: First, whether precursors of allicin and/or its degradative products may contribute to the relaxation. Second, the mechanisms of allicin-induced NO-dependent relaxation. Third, the mechanisms of allicin induced direct vascular smooth muscle relaxation. Fourth, the chemical/biological fate of allicin following its entry into the blood circulation, which may explain the transient vasodepressor responses in intact animals. And Fifth, if oral administration of garlic with different allicin yields produces a graded protection against hypoxia and monocrotaline-induced pulmonary hypertension. We believe that our novel HPLC/MS-MRM method of detection/quantitation of allicin coupled with a comprehensive approach to studying its biological activities both in vitro and in vivo will provide important new insights into the role of garlic/allicin modulation of pulmonary vasoactivity and function. Our long-term objective is to identify novel therapeutic agents to improve the clinical management of pulmonary vascular disorders. [unreadable] [unreadable] [unreadable]